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The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis. more...

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5′ untranslated region (UTR). Differential 5′ UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5′ UTR lengthened by over 100 bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency. [ABSTRACT FROM AUTHOR] more...

Simple Summary: Crosstalk between our cells and the microbes within our bodies has long been known to play a role in normal physiology and disruption of this crosstalk can lead to pathological conditions. We recently provided evidence that the microbes within lung cancers are enriched for methionine-producing constituents. Cancer cells can utilize this methionine under conditions of limiting nutrients. Herein, we expand these studies to demonstrate that biomolecules produced by bacteria can influence multiple signaling and metabolic pathways within lung cancer cells. By furthering our understanding of how microbes can influence tumorigenic and metastatic processes, we may be able to design novel therapeutics for treating lung cancer. Introduction: Recent studies have discovered that lung cancer subtypes possess distinct microbiome profiles within their tumor microenvironment. Additionally, the tumor-associated microbiome exhibits altered bacterial pathways, suggesting that certain bacterial families are more capable of facilitating tumor progression than others. We hypothesize that there exists a crosstalk between lung adenocarcinoma (LUAD) cells and bacterial cells. Methods and Materials: RNA sequencing (RNA-seq) was performed on LUAD cell lines to explore the paracrine signaling effects of bacterial biomolecules. Based on our RNA-seq data, we investigated glycolysis by measuring glucose uptake and lactate production, invasive potential through invasion assays, and epithelial-to-mesenchymal transition (EMT) markers. Since lipopolysaccharides (LPS), abundant on the cell walls of Gram-negative bacteria, can activate toll-like receptor 4 (TLR4), we inhibited TLR4 with C34 to assess its relationship with the observed phenotypic changes. To identify the bacterial biomolecules responsible for these changes, we treated the media with RNAse enzyme, charcoal or dialyzed away molecules larger than 3 kDa. Results and Discussion: RNA-seq revealed 948 genes upregulated in the presence of E. coli biomolecules. Among these, we observed increased expression of Hexokinase II (HKII), JUN proto-oncogene, and Snail Family Transcriptional Repressor 1. We verified the elevation of glycolytic enzymes through Western blot and saw elevation of 2-deoxyglucose uptake and lactate production in LUAD cell lines incubated in E. coli biomolecules. In addition to E. coli elevating glycolysis in LUAD cell lines, E. coli exposure enhanced invasive potential as demonstrated by Boyden chamber assays. Notably, inhibition of TLR4 did not reduce the impact of E. coli biomolecules on glycolysis or the invasive potential of LUAD. Modulating the E. coli-supplemented media with RNAse enzyme or dextran-coated charcoal or using a spin column to remove biomolecules smaller than 3 kDa resulted in changes in HKII and Claudin protein expression. These findings suggest a direct relationship between E. coli and LUAD, wherein several cancer hallmarks are upregulated. Future studies should further investigate these bacterial biomolecules and their role in the tumor microenvironment to fully understand the impact of microbial shifts on cancer progression. [ABSTRACT FROM AUTHOR] more...

Inflammation and infection are key promoters of colon cancer but the molecular interplay between these events is largely unknown. Mice deficient in leukotriene B4 receptor1 (BLT1) are protected in inflammatory disease models of arthritis, asthma and atherosclerosis. In this study, we show that BLT1-/- mice when bred onto a spontaneous tumor (ApcMin/+) model displayed an increase in the rate of intestinal tumor development and mortality. A paradoxical increase in inflammation in the tumors from the BLT1-/-ApcMin/+ mice is coincidental with defective host response to infection. Germ-free BLT1-/-ApcMin/+ mice are free from colon tumors that reappeared upon fecal transplantation. Analysis of microbiota showed defective host response in BLT1-/- ApcMin/+ mice reshapes the gut microbiota to promote colon tumor development. The BLT1-/-MyD88-/- double deficient mice are susceptible to lethal neonatal infections. Broad-spectrum antibiotic treatment eliminated neonatal lethality in BLT1-/-MyD88-/- mice and the BLT1-/-MyD88-/-ApcMin+ mice are protected from colon tumor development. These results identify a novel interplay between the Toll-like receptor mediated microbial sensing mechanisms and BLT1-mediated host response in the control of colon tumor development. more...

Multiple Sclerosis (MS) is an autoimmune neurodegenerative disease affecting approximately 3 million people globally. Despite rigorous research on MS, aspects of its development and progression remain unclear. We utilized a publicly available RNA-seq dataset (GSE138614) consisting of the post-mortem white matter tissues of five donors without any neurological disorders and ten MS patient donors. We investigated gene expression levels correlated with tissue inflammation and alternative splicing to identify possible pathological isoforms in MS tissues. We identified RNA-binding motifs, differentially expressed RNA-binding proteins, and single-nucleotide polymorphisms (SNPs) to unravel possible mechanisms of alternative splicing. Genes with expression changes that were positively correlated with tissue inflammation were enriched in the immune system and receptor interaction pathways. Genes showing a negative correlation were enriched in nervous system development and in metabolic pathways. A comparison of normal-appearing white matter (NAWM) and active or chronic active lesions within the same donors identified genes playing roles in immunity, white matter injury repair, and remyelination. We identified exon skipping events and spontaneous SNPs in membrane-associated ring-CH-type finger-1 (MARCHF1), UDP glycosyltransferase-8 (UGT8), and other genes important in autoimmunity and neurodegeneration. Overall, we identified unique genes, pathways, and novel splicing events that can be further investigated as potential novel drug targets for MS treatment. [ABSTRACT FROM AUTHOR] more...

RNA-binding proteins (RBPs) play a critical role in the regulation of alternative splicing (AS), a prevalent mechanism for generating transcriptomic and proteomic diversity in eukaryotic cells. Studies have shown that AS can be regulated by RBPs in a binding-site-position dependent manner. Depending on where RBPs bind, splicing of an alternative exon can be enhanced or suppressed. Therefore, spatial analyses of RBP motifs and binding sites around alternative exons will help elucidate splicing regulation by RBPs. The development of high-throughput sequencing technologies has allowed transcriptome-wide analyses of AS and RBP-RNA interactions. Given a set of differentially regulated alternative exons obtained from RNA sequencing (RNA-seq) experiments, the rMAPS web server (http://rmaps.cecsresearch.org) performs motif analyses of RBPs in the vicinity of alternatively spliced exons and creates RNA maps that depict the spatial patterns of RBP motifs. Similarly, rMAPS can also perform spatial analyses of RBP-RNA binding sites identified by cross-linking immunoprecipitation sequencing (CLIP-seq) experiments. We anticipate rMAPS will be a useful tool for elucidating RBP regulation of alternative exon splicing using high-throughput sequencing data. more...

Introduction Lung cancer remains the leading cause of cancer-related deaths worldwide, accounting for approximate 2.1 million new cases and 1.7 million deaths annually (1). Although cancer immunotherapy has been proclaimed [...], Macrophages have been linked to tumor initiation, progression, metastasis, and treatment resistance. However, the transcriptional regulation of macrophages driving the protumor function remains elusive. Here, we demonstrate that the transcription factor c-Maf is a critical controller for immunosuppressive macrophage polarization and function in cancer. c-Maf controls many M2-related genes and has direct binding sites within a conserved noncoding sequence of the Csf-1r gene and promotes M2-like macrophage-mediated T cell suppression and tumor progression. c-Maf also serves as a metabolic checkpoint regulating the TCA cycle and UDP-GlcNAc biosynthesis, thus promoting M2-like macrophage polarization and activation. Additionally, c-Maf is highly expressed in tumor-associated macrophages (TAMs) and regulates TAM immunosuppressive function. Deletion of c-Maf specifically in myeloid cells results in reduced tumor burden with enhanced antitumor T cell immunity. Inhibition of c-Maf partly overcomes resistance to anti-PD-1 therapy in a subcutaneous LLC tumor model. Similarly, c-Maf is expressed in human M2 and tumor-infiltrating macrophages/monocytes as well as circulating monocytes of human non-small cell lung carcinoma (NSCLC) patients and critically regulates their immunosuppressive activity. The natural compound [beta]-glucan downregulates c-Maf expression on macrophages, leading to enhanced antitumor immunity in mice. These findings establish a paradigm for immunosuppressive macrophage polarization and transcriptional regulation by c-Maf and suggest that c-Maf is a potential target for effective tumor immunotherapy. more...

Spinal cord injury (SCI) is a debilitating condition with an estimated 18,000 new cases annually in the United States. The field has accepted and adopted standardized databases such as the Open Data Commons for Spinal Cord Injury (ODC-SCI) to aid in broader analyses, but these currently lack high-throughput data despite the availability of nearly 6000 samples from over 90 studies available in the Sequence Read Archive. This limits the potential for large datasets to enhance our understanding of SCI-related mechanisms at the molecular and cellular level. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies, as well as homologous discovery across species. We have processed 1196 publicly available RNA-Seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments. [ABSTRACT FROM AUTHOR] more...